. Cryptococcus neoformans remains a significant cause of morbidity and mortality in patients with AIDS and other immunosuppressive states. Despite advances in the treatment of cryptococcal meningitis, there is still no curative treatment in AIDS patients. In the past five years, there has been significant advances in the molecular biology of C. neoformans. Over a dozen genes have been cloned and characterized, transformation systems have been developed, and pioneering studies on virulence-related genes have been described. Overall, these studies have shown that not only is this organism an important field of study because of its relevance to human health, but it provides an ideal model system of fungal pathogenesis. The focus of the Edman laboratory, which has been an understanding of the mechanisms of dimorphism in C. neoformans, the characterization of the mating type loci, and the role of the MAT alpha locus in promoting virulence, will be continued through studies described in this application. Recent observations by Dr. Edman have demonstrated a true yeast to hyphae dimorphic transition in C. neoformans wild type, haploid, MAT alpha cells. The control of this transition will be characterized by the isolation of mutants in this pathway, cloning the genes defined by these mutants, and morphologic assessment of the hyphae produced in mutant and wild type strains. The mating type loci of C. neoformans will be characterized by transcript analysis, DNA sequence, and gene disruption. While there are over a dozen genes within these complex loci, two MATalpha genes that have already been isolated, the pheromone gene and a homologue of the S. cerevisiae PRT1 gene, will receive initial scrutiny. The mature pheromone will be characterized by purification from overexpressing cells and its structure determined by mass spectrometry. Cis- and trans-acting factors responsible for pheromone gene regulation will be identified, isolated and characterized. These studies will be complemented by the continuing isolation of C. neoformans mutants that are unable to form filaments after mating. MATalpha deletion and MATalpha::MATalpha replacement strains will be constructed to formally test the hypothesis that there are virulence determinants within the MATalpha locus. These strains should provide new insights into the mechanisms of pathogenesis of C. neoformans.